1. Investigating the relationship between serum 11-deoxycortisol levels and blood pressure Helen Turner MSc Project

2. Do levels of 11-deoxycortisol affect blood pressure in a normal population? Objectives: To optimise and validate a novel method for the measurement of serum 11-deoxycortisol. To establish the relationship between serum 11-deoxycortisol and blood pressure in an adult population.

3. Hypertension Without treatment increased risk of cardiovascular and renal complications Can be split into essential or secondary hypertension 95% of patients have essential hypertension which can be attributed to both environmental and genetic factors 5% have secondary hypertension Where the cause is known, and if treated will alleviate the symptoms

4. Steroids & Hypertension Supportive evidence –Abnormal steroid production can lead to hypertension-e.g in PA, CAH, Cushings & GSH –Male presenting with hypertension, may have elevated glucocorticoid metabolites in their urine –Alterations in the 11-deoxycortisol: cortisol ratio has been seen in hypertensive patients

5. Cholesterol 17-OH PregnenoloneDHEA Aldosterone 18-OH Corticosterone Corticosterone Deoxycorticosterone Progesterone Pregnenolone Androstenedione Cortisol 17-OH Progesterone 11-Deoxycortisol 11ß-Hydroxylase 21ß-Hydroxylase

6. 11-deoxycortisol and 11ß hydroxylase Mutations in CYB11B1 alter 11ß hydroxylase activity 11ß hydroxylase deficient patients have increased levels of 11-deoxycortisol Present with hypertension Therefore… could slight changes in 11ß hydroxylase activity, cause slight variations in blood pressure?

7. Method Extraction –400ul sample + 100ul assay buffer, add to 2.5mls CCL 4 –Discard aqueous layer Drying down –1ml extract dried under low pressure N 2 @37ªC Reconstitution –With 200ul assay buffer, @RT for 30mins DELFIA – Dissociation enhanced lanthanide fluoroimmunoassay

8. DELFIA Set up Patient Sample Plate Wall Coating Antibody Highly fluorescent Eu- (2-NTA) 3 (TOPO) 2-3 Enhancement Solution Anti 11- deoxycortisol Biotinylated 11- deoxycortiol Eu 3+ labelled Avidin

9. Validation Recovery 113% Cross reactivity 11% 17OHP <0.1 other steroids Lower limit of Detection 0.9nmol/L Precision within assay 16% between assay20% Comparison to RIAr=0.72 p=<0.0001

10. Materials Patient samples From MRC Hertfordshire study 271 patients: 140males and 131 females Born between 1931 and 1939 in Hertfordshire No major medical conditions Clinics between June 2003 and June 2004 Blood Pressure Recorded at clinics Lying and seated positions

11. Results: Females r=-0.01, p=0.8837 r=0.01, p=0.9348

12. Results: Males r=-0.01, p=0.2280 r=0.01, p=0.8949

13. Conclusions We produced a simple method for measuring 11-deoxycortisol in serum There is no relationship between 11- deoxycortisol and blood pressure in a normal adult population

14. Future Work Measure cortisol To compare 11deoxycortisol in the hypertensive patient to normal patients Measure 11-deoxycortisol in a greater age range

15. Acknowledgements Pete Wood Christine Glenn MRC- Southampton Karen Jameson Martyn Egerton (Epsom and St Helier Hosptial)

17. Cross Reactivity SteroidCross reactivity (%) Cortisone0.37 Progesterone0.6 Prednisone0.39 21-deoxycortisol<0.1 Aldosterone<0.1 Adione<0.1 Cortisol0.6 17 OHP14%

18. Precision Low QCMedium QC High QC Mean (nmol/L) 2.412.956.5 SD (nmol/L) 0.52.17.5 %CV20.416.313.2 Low QCMedium QC High QC Mean (nmol/L) 4.8332.8556.44 SD (nmol/L) 1.364.709.31 %CV31.014.3116.50 Intra Assay Variation Inter Assay Variation

19. Hypertension StageSystolic BP (mmHg) Diastolic BP (mmHg) Normal<120<80 Pre- hypertensive 120-13980-89 Stage 1140-15990-99 Stage 2160>100> 6 th report of the Joint National Committee on the Prevention, Detection, Evaluation and treatment of Blood Pressure

20. Steroids & Hypertension 2 Non- supportive evidence: –No significant correlation between cortisol levels in plasma and the degree of hypertension –Deoxycorticosterone levels shows no correlation with blood pressure –The metabolites of deoxycorticosterone, are potent mineralocorticoids but require 11- hydroxylase for their production

21. Lower Limit of Detection 0.9nmol/L Recovery Mean recovery 113%